荧光原位杂交技术分析人结肠菌群方法研究

建立荧光原位杂交技术分析人体内结肠菌群的方法。取受试者新鲜粪便 ,选用 5种特异性的 16SrRNA寡核苷酸探针 ,检测粪便样本收集后的保存时间、温度 ,离心条件及样本固定液存放时间对杂交计数结果的影响。结果建立最佳实验条件为 :粪便样本收集后应尽快在 4℃下保存 ,放置时间不要超过 12小时即作处理 ;样本的适宜离心条件为 70 0g 2分钟 ;样本用多聚甲醛固定后在 - 80℃下存放时间不要超过 5个月。该方法具有较好的稳定性 ,可以有效地检出个体之间结肠菌群的差异。     

Patterns of CD16 and CD56 expression in persistent expansions of CD3+NKa+ lymphocytes are predictive for clonal T-cell receptor gene rearrangements

Phenotypic characteristics, and correlations between the expression of membrane NK-associated (NKa) determinants (CD11b, CD16, CD56 and CD57) and T cell receptor (TCR) genotypic patterns, were examined in 25 patients with persistent (greater than 6 months) expansions of CD3+WT31+NKa+ (CD8+ and CD8dim+) lymphocytes. These studies showed that distinct NKa phenotypic profiles were restricted to cases with rearranged TCR configurations and that clonal CD3+NKa+ components could be predicted in most cases by assessing relationships between membrane CD16 and CD56 expression. For all normal NKa subpopulations, there was a high correlation (P less than 0.0001; n = 31) between the expression of these two membrane determinants. Markedly increased CD16 expression by CD3+NKa+ cells, in relation to CD56 (i.e. a high CD16:CD56 ratio), was found exclusively in cases with rearranged TCR (13/16 cases); 2/3 of the remaining cases showing significantly reduced CD16:CD56 ratios and high (greater than 2.0) CD3+CD56+ absolute numbers. In contrast, 7/9 of the germline TCR cases had a normal CD16:CD56 ratio and 2/9 a decreased ratio with low (less than 1.0) CD3+CD56+ absolute numbers. A high ratio of CD16:CD56 expression by CD3+NKa+ lymphocytes was therefore informative for 82% of TCR rearrangements in this series; and analysis of CD16 and CD56 expression was predictive for germline and rearranged TCR configurations in 24/25 persistent CD3+NKa+ expansions.